Non-small cell lung cancer (NSCLC) is a highly aggressive form of malignancy that is associated with metastasis and poor response to chemotherapy. Non-small cell lung cancer cells exhibit high resistance to undergo apoptosis, at least in part due to a disabled apoptotic machinery. In particular, partial defects in the caspase-dependent pathway in NSCLC cells conferred resistance to apoptosis induced by DNA-damaging agents such as etoposide or 3-radiation (9,14). Hence, a major therapeutic avenue in combating the aggressiveness and chemotherapy resistance of NSCLC is through effective induction of apoptosis by the alternative caspase-independent cell death pathway. The goal of this project is to address the critical barrier to NSCLC treatment by obtaining the information needed to understand the role of the novel caspase- independent anoikis effector, Bcl2-inhibitor of transcription (Bit1), in the apoptosis resistance and malignant transformation of NSCLC cells. Several observations indicate the nonfunctionality of the Bit1 pathway in lung cancer: i) Bit1 expression is significantly suppressed in various types of NSCLC tissue relative to counterpart normal lung tissue (Fig. 1A and 1B) and ii) an inhibitor of Bit1 function, TLE1, functions as a lung specific oncogene and is overexpressed in human lung tumors (1). While ectopic expression of cytoplasmic localized Bit1 in the NSCLC cell line NCI-H460 induces apoptosis (Fig 1D-F), restoration of mitochondrial localized Bit1 in H460 cells enhances anoikis sensitivity and attenuates anchorage independent growth and resistance to etoposide-induced apoptosis (Fig. 2). Based on the published data and our preliminary results, our objective is to test the hypothesis that malignant NSCLC cells are likely to bypass the Bit1 apoptotic pathway to become anoikis resistant and anchorage-independent, and that activation of the Bit1 pathway may be effective in inducing apoptosis and attenuating the aggressive phenotypes in NSCLC cells. The specific aims are: 1) to examine the regulation and relevance of the Bit1 apoptosis pathway in the survival and apoptotic resistance of NSCLC cells, 2) to test the hypothesis that the Bit1 apoptotic pathway regulates the aggressive phenotypes of NSCLC cells by alteration of mitochondrial Bit1 expression and its impact on anoikis resistance, anchorage- independent growth potential, or chemoresistance of NSCLC cells, and 3) to understand and dissect the apoptotic machinery in NSCLC using Bit1 as a tool. The outcome of this study will delineate the importance of the suppression or blockage of the Bit1 apoptotic pathway in the acquisition of anoikis resistance and anchorage-independence by the NSCLC. We believe that activation of the caspase-independent cell death pathway via the Bit1 apoptotic function will result in a successful eradication of chemoresistance in NSCLC. Thus, Bit-1 may serve as a novel therapeutic target for the treatment of this aggressive cancer. PHS398 (Rev. 5/01) Page Continuation Format Page PUBLIC HEALTH RELEVANCE: A Role of Bit1 in the Apoptosis Resistance, Anoikis Insensitivity, and Chemoresistance in Non-small cell lung cancer (NSCLC) Cells Non-small cell lung cancer (NSCLC) is a highly aggressive disease with poor prognosis and high mortality. The dismal prognosis is in part due to its resistance to conventional chemotherapy and its propensity to disseminate to distant organs. To circumvent the aggressiveness and chemoresistance of NSCLC, it is imperative to identify novel cell death pathway(s) that may enhance the apoptotic sensitivity of NSCLC cells.